Nitric oxide (NO) is widely used by surgeons, anesthesiologists and critical care physicians managing critically-ill patients (e.g., inhaled NO for treatment of pulmonary hypertension and nitrovasodilators for decreasing afterload and for improving myocardial perfusion). Tolerance to these NO-based therapies occurs clinically and the extent to which this NO-induced NO hyporesponsiveness is due to decreased activity in the vascular smooth muscle NO-cGMP-cGK signaling system is unknown. The objective of this proposal is to determine the mechanisms by which chronic exposure to NO affects this signaling system in vascular smooth muscle. Two broad categories of mechanism for the development of chronic NO-induced NO hyporesponsiveness involving the vascular smooth muscle NO- cGMP-cGK signaling system are possible: 1) changes in [cGMP]i regulation and 2) reductions in the sensitivity to cGMP (cGMP sensitivity). To explore these mechanisms, we will utilize a cultured pulmonary artery preparation (PA) which permits experiments in fully functional, differentiated vascular smooth muscle cells. This cultured vessel preparation makes it possible to directly relate chronic NO-induced changes in the vascular smooth muscle NO-cGMP-cGK signaling system to chronic NO-induced decreases in NO responsiveness. Chronic NO exposure will be achieved using both NO-donors, and endogenously-derived NO following nitric oxide synthase gene transfer. Greater insight into the factors responsible for chronic NO-induced NO hyporesponsiveness will aid in the development of future NO-based therapeutic strategies. This is of particular relevance in the pulmonary circulation, in which inhaled NO is administered as a therapy for pulmonary hypertension, yet a large fraction of these patients are unresponsive to the NO. Methods used in the proposed studies include isometric force measurements, RT-PCR to isolate mRNA for all soluble guanylyl cyclase subunits (sGC) and cGMP-dependent protein kinase (cGK) isoforms, quantitative RT-PCR and immunoblotting to measure expression of each, and sGC and cGK enzyme activity measurement. We will use a combination of pharmacological probes and novel antisense oligomers to determine the mechanism by which chronic NO treatment decreases the expression and activity of sGC subunits and cGK isoforms.